Monomeric red fluorescent proteins

ABSTRACT

Disclosed are sequences encoding monomeric variants of DsRed fluorescent proteins and methods of use.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application No.60/560,340, filed Apr. 7, 2004, which is incorporated by reference inits entirety.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

This invention was made with government support under MCB-9875939,awarded by the National Science Foundation, and under RPG-00-245-01-CSM,awarded by the American Cancer Society. The government has certainrights in the invention.

INTRODUCTION

Fluorescent proteins such as green fluorescent protein (GFP) areinvaluable tools used in pure and applied research. Although fluorescentproteins have been widely available for a relatively short time, theyhave had an important impact on biomedical research, contributing to ourunderstanding of basic cellular and developmental processes thatunderlie health and disease. GFP and its relatives are widely used formedically-oriented research. For example, GFP has been used to analyzebacterial gene expression during infection, to visualize tumor cellbehavior during metastasis, and to monitor GFP fusion proteins in genetherapy studies. Fluorescent proteins are also useful in high-throughputscreens for drug discovery.

A red fluorescent protein produced by the coral Discosoma and designatedDsRed (wild-type DsRed) is potentially useful as a fluorescent reporterprotein or as a fusion tag. A red fluorescent protein is particularlyattractive because of its suitability for use in conjunction withfluorescent proteins having different fluorescent properties, such asGFP. However, wild-type DsRed suffers from certain drawbacks.

First, the maturation process that yields the red fluorophore is slow,with a half-time of ˜12 h at 37° C. Second, wild-type DsRed occurs as astable tetramer of four very similar polypeptides, which makes its useas a fluorescent reporter in a fusion protein problematic. For example,tetramerization of the DsRed fusion protein may interfere with orperturb the function or localization of the protein. In addition, DsRedtetramers undergo higher-order aggregation. Fusion of DsRed to membraneproteins or to oligomeric proteins often produces large aggregates.Efforts to develop variants of DsRed having a reduced tendency to formtetramers have met with limited success, in that the variants sufferfrom disadvantages such as undesirable shifts in fluorescence or morerapid photobleaching.

There is, therefore, ongoing interest in developing new fluorescentprotein labels with improved characteristics as experimental andclinical tools.

SUMMARY OF THE INVENTION

The present invention provides polynucleotide encoding a variantpolypeptide of wild-type DsRed or the rapidly maturing DsRed.T4, atetrameric variant that has the substitutions described herein below.The variant polypeptide has reduced oligomerization relative wild-typeDsRed and has a fluorescence spectra similar to the fluorescence spectraof the wild-type DsRed, the variant polypeptide comprising the aminoacid substitutions K83M, K163H, and Y193H and further comprises at leastof one amino acid substitutions E26Y, K92T, V96S, T106E, T108Q, 1125K,S131A, I180V, and M182K.

In another aspect, the invention provides isolated polynucleotidesencoding a variant polypeptide of the rapidly DsRed.T4 and exhibitingreduced oligomerization relative to DsRed.T4 and detectable redfluorescence, the variant polypeptide comprising at least one of aminosubstitutions K83M or K83L; K163Q, K163M, or K163H; and Y193H, andfurther comprising at least three amino acid substitutions selected fromE26Y, K92T, V96S, T106E, T108Q, I125K, S131A, I180V, and M182K; andfurther comprising at least three amino acid substitutions selected fromthe group consisting of R149K, R153Q, H162S, L174T, E176D, Y192N, R216H,H222S, L223G, and F224S.

In other aspects, the invention provides genetic constructs comprisingthe polynucleotides, vectors comprising the constructs, cells comprisingthe constructs, variant polypeptides encoded by the polynucleotides, andmethods of obtaining expression of the polynucleotides.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts the hydrophobic interface (FIG. 1A) and the polarinterface (1B) involved in tetramerization of DsRed, the latter of whichalso includes the hydrophobic tail (residues 222–225).

FIG. 2 compares the excitation and emission spectra for a derivative oftetrameric variant DsRed.T4 having a K83M substitution, and a derivativeof DsRed.T4 having K83M, K163H, and Y193H substitutions.

DETAILED DESCRIPTION

The present invention provides polypeptide variants of wild-typeDiscosoma sp. red fluorescent protein (DsRed). Wild-type DsRed formsoligomers (e.g., dimers or tetramers) under physiologic conditions,which, in the native polypeptide, appears to play a role in itsfluorescence activity. A coding sequence for wild-type Discosoma sp. redfluorescent protein (DsRed) is shown in SEQ ID NO:1, and the amino acidsequence of DsRed is shown in SEQ ID NO:2. The polypeptide variants ofthe present invention have a reduced tendency to oligomerize relative towild-type DsRed. This reduced tendency to oligomerize may be assessed byany suitable means, whether in vivo or in vitro, as described below.

Polypeptide variants were genetically engineered, as described in detailbelow, by altering a sequence encoding a rapidly maturing tetramericvariant of wild-type DsRed, designated DsRed.T4 (SEQ ID NO:3). DsRed.T4is described in further detail in co-pending U.S. patent applicationSer. No. 10/844,064, which is incorporated by reference in its entirety.Relative to the wild-type DsRed of SEQ ID NO:2, DsRed.T4 contains thefollowing substitutions:

P(-4)L H41T R2A N42Q K5E V44A N6D A145P T21S T217A

Relative to either wild-type DsRed or DsRedT4, the polypeptides of thepresent invention exhibit a reduced tendency to oligomerize or formtetramers, and exist primarily as monomers under physiologic conditions.The polypeptide variants exhibit detectable red fluorescence. By“detectable red fluorescence” it is meant that the fluorescence overlapsthe emission spectra of wild-type DsRed is distinguishable overbackground. Preferably, the emission spectra is similar to that ofwild-type DsRed.

To develop monomeric DsRed variants, a polynucleotide sequence encodingDsRedT4 was modified using the following general approach. As oneskilled in the art will appreciate, one could also begin with apolynucleotide encoding another DsRed, for example, wild-type DsRed.

Mutations were introduced into a polynucleotide sequence encodingDsRedT4 to disrupt the tetramerization interfaces that form betweenDsRed polypeptides. Amino acids suspected of contributing tooligomerization were replaced by amino acids that are likely to reduceoligomerization.

Crystal structures of DsRed reveal residues that may be involved intetramerization. The region that we have designated the “hydrophobicinterface” primarily engages in hydrophobic interactions that excludethe solvent (FIG. 1A). The region that we have designated the “polarinterface” primarily engages in polar interactions, although thisinterface also includes some hydrophobic residues (Leu-174 and theC-terminal tail) that interact with the opposing subunit (FIG. 1B).

Two considerations guided selection of residues to be mutated and thespecific substitutions to be tested. First, certain residues are morelikely than others to make a significant energetic contribution tooligomerization. For example, a statistical study revealed thatisoleucine side chains are often important for protein-proteininteractions (Bogan and Thorn, 1998). In designing the variants of thepresent invention, it was assumed that surface-exposed hydrophobicresidues are likely to promote oligomerization, and therefore, suchresidues were considered prime candidates for mutagenesis. Thus, it islikely that generation of a stable monomer requires modification ofresidues Ile-180 and Ile-125 (FIG. 1A). Second, the DsRed protein willtolerate substitutions more readily at some positions than at others.Many mutations at the tetramerization interfaces will have secondaryeffects on the folding and/or maturation of the protein. To makeeducated guesses about which residues can be mutated and whichalternative residues can be substituted, we used sequence alignmentsbetween DsRed, GFP, and the other known fluorescent proteins (Matz etal., 1999; Wall et al., 2000).

In some polypeptide variants, mutations in two of the tetramerizationinterfaces resulted in a loss of tetramerization, which can be measuredin vitro or in vivo as described in the Examples, and loss offluorescence. It was, therefore, speculated that tetramerization may beimportant for fluorescence in wild-type DsRed, possibly because theautocatalytic formation of the chromophore may stabilize the foldedstructure of DsRed or because oligomerization may increase the quantumyield.

To restore fluorescence to an oligomerization-disrupted or monomericDsRed variants, site-directed mutagenesis was performed to introducespecific changes that were expected to stabilize the immature and/ormature forms of the protein. Two general classes of mutations wereintroduced in an attempt to restore fluorescence after disrupting thetetramer: (1) mutations that may stabilize folding intermediates topermit the protein to mature even in the absence of oligomerization; and(2) mutations expected to rigidify mature DsRed to enhance quantumyield.

In a second strategy to restore fluorescence to anoligomerization-disrupted or monomeric DsRed variant, a wide range ofmutations were introduced into either the entire DsRed coding sequenceor into a selected segment of the coding sequence. The variants thusgenerated were screened for improved fluorescence.

The effect of various mutations has, in some cases, been demonstrated,and in other cases, has been presumed or inferred. The mutations can bedivided into the following six categories of effects:

-   1. Mutations that presumably stabilize immature folding    intermediates. In tetrameric DsRed variants, K83 and K163 face the    interior of the protein. The presence of these highly polar internal    residues is likely to destabilize folding intermediates. Therefore,    K83M and K163H substitutions were introduced to reduce the polarity    at these positions. The K163H substitution is relatively    conservative because histidine is still somewhat polar. The K83M    substitution introduces a hydrophobic residue, and red-shifts the    fluorescence spectra. However, it was discovered by random    mutagenesis that a Y193H substitution reverses the spectral changes    caused by K83M, and also enhances brightness. Residue 193 is close    to residue 83 of the folded protein, so the Y193H substitution may    compensate for the reduced polarity caused by K83M. Variants    containing the trio of substitutions K83M, K163H, Y193H seem to be    stabilized, as indicated by their ability to tolerate interface    mutations that are not tolerated by DsRed.T4.-   2. Mutations that disrupt the hydrophobic tetramerization interface.    This category comprises E26Y, K92T, V96S, T106E, T108Q, I125K,    S131A, I180V, and M182K, each of which is predicted to disrupt    intersubunit interactions at the hydrophobic interface.-   3. Mutations that disrupt the polar tetramerization interface. This    category comprises R149K, R153Q, H162S, L174T, E176D, Y192N, R216H,    H222S, L223G, F224S, and L225Q, each of which is predicted to    disrupt intersubunit interactions at the polar interface. The    hydrophobic C-terminal tail of DsRed is considered to be part of the    polar interface.-   4. Mutations that enhance monomer fluorescence, presumably by    stabilizing and/or rigidifying the protein. This category comprises    V71A, C117T, V175C, S179T, S203N, and G219A. Note that C117T also    eliminates a surface cysteine residue that might otherwise be    oxidized within the secretory pathway.-   5. Mutations that improve expression in E. coli. When a variant such    as DsRed.T4 is produced in E. coli using its own start codon, the    protein levels are much lower than when the same protein is produced    using an N-terminal hexahistidine tag, which suggests that the 5′    end of the gene is important for expression in bacteria. The coding    sequences were altered such that the putative translation products    contained substitutions in residues 2–4 and screened for strong    expression in E. coli. A polypeptide having the substitutions A2D,    S3N, and S4T was found to be strongly expressed. These N-terminal    mutations probably enhance translation in E. coli, an effect that    may not occur with expression in eukaryotic cells. In addition, the    N-terminal mutations might have some beneficial effect on the    maturation and/or stability of DsRed.-   6. Mutations that reduce the general “stickiness” of the protein. We    introduced a number of surface mutations that lie outside of the    tetramerization interfaces that should reduce the tendency of the    protein to adhere to other macromolecules. Specifically, we targeted    basic residues (especially arginines) and large, hydrophobic    residues. The substitutions in this category include R13Q, R36K,    K47Q, M141A, and I210V. We have shown that these substitutions do    not significantly reduce fluorescence, and expect that variants    containing one or more of these substitutions may exhibit reduced    aggregation with other macromolecules. Reduced aggregation may be    evaluated by comparing the solubility of DsRed variants to that of    wild-type DsRed.

An example of a DsRed monomeric variant containing numerous mutationsand identified as DsRed.M1 (SEQ ID NO:5) is described below. Of course,useful monomeric variants of the presently claimed invention havingfewer mutations than those contained in the DsRed.M1 may be developedusing the guidance and teaching herein of this disclosure.

DsRed.M1 was further modified by introducing D6N, a reversion to thenative sequence, in order to reduce the number of acidic residues at theN-terminus. The additional substitutions K121H, K168E, D169G, D115G, andG116N were made to improve brightness or fluorescence.

Once a DsRed monomeric variant having desirable characteristics isidentified, one may, of course, use any polynucleotide sequence encodingthe variant to express the variant. For example, the polynucleotideencoding the variant may be modified for optimal expression in aparticular organism in view of the preferred codon usage of thatorganism. The polynucleotide may be operably linked to an inducible orconstituitive promoter functional in the intended cell or organism. Thepolynucleotide may be linked in-fram to a second polynucleotide sequenceencoding a polypeptide of interest to form a sequence encoding a fusionprotein in which the polypeptide of interest is labeled with the DsRedmonomeric variant at its N- or C-terminus.

EXAMPLES

Selection of Amino Acid Substitutions to Reduce Tetramerization

Site-directed mutagenesis using standard methods well-known to one ofordinary skill in the art was used to disrupt the two tetramerizationinterfaces of DsRed. Various substitutions at each position were made,and fluorescence was evaluated as described below.

The Hydrophobic Interface

Extensive mutagenesis of the hydrophobic interface was performed,beginning with the DsRed.T4 variant. DsRed.T4 contains a T21Ssubstitution. In addition, Met-182, Ile-180, Val-96 and Ile-125 weresubstituted with more polar residues, and a hydrogen-bonding residue wasremoved through an S131A substitution. The DsRed.T4 variant having thesesubstitutions was designated DsRed.D1. This variant is probably dimeric.An additional V104A substitution is tolerated in the DsRed.D1background. It is of note that, in the related fluorescent proteinHcRed, a single Leu-to-His mutation in the putative hydrophobicinterface at the position corresponding to Ile-125 of DsRed wasreportedly sufficient to convert HcRed into a dimer (Gurskaya et al.,2001). Bacterial colonies producing DsRed.D1 are somewhat lessfluorescent than those producing DsRed.T4, but the signal with DsRed.D1is still strong.

The Polar Interface

Initial attempts to mutagenize the polar interface yielded reduced thefluorescence. For example, Leu-174 forms hydrophobic interactions withthe opposing subunit, but all of the Leu-174 substitutions that weinitially tried, including a conservative change to Val, virtuallyabolished fluorescence. It was thus concluded that Leu-174 is requiredfor fluorescence and should not be mutated. Similar results wereobtained with His-162, which associates with its counterpart on theopposing subunit in an unusual stacking interaction, and with His-222,which inserts into a groove in the opposing subunit. Conservativemutations of these His residues to Ser or Asn severely diminished thefluorescence. Indeed, mutagenesis of most the key residues at the polarinterface were found to impair fluorescence.

Assessing Oligomeric State of DsRed Variants

The oligomeric state of a DsRed variant may be assessed by nondenaturingSDS-PAGE or size exclusion chromatography.

For nondenaturing SDS-PAGE, one μg of each purified DsRed variant orwild-type DsRed was mixed with SDS-containing sample buffer on ice andimmediately electrophoresed at 4° C. in a 10% polyacrylamide gel,followed by staining with Coomassie Blue. Additional aliquots of ofwild-type DsRed and DsRed variants were denatured by boiling prior toelectrophoresis.

Gel filtration chromatography may be used to indicate whether afluorescent protein exists predominantly as a monomer, dimer or tetramer(Gurskaya et al., 2001). Optionally, gel filtration will be conductedusing the Pharmacia FPLC system. An extension of this method known assmall-zone size-exclusion gel filtration chromatography could be used tomeasure association constants (Raffen and Stevens, 1999).

Oligomerization states and association constants of our DsRed variantsmay be evaluated using analytical ultracentrifugation (Laue andStafford, 1999). This approach was used by Baird et al. (2000) for theirinitial demonstration that DsRed is a tetramer. A Beckman XL-Aanalytical ultracentrifuge will be used. Velocity sedimentation will beused to ascertain which DsRed species are present (monomers, dimersand/or tetramers), and then equilibrium sedimentation will be used tomeasure the oligomer association constants (Laue and Stafford, 1999).

Analytical ultracentrifugation will be carried out in collaboration withBorries Demeler (University of Texas Health Sciences Center at SanAntonio}, who has extensive experience with analyticalultracentrifugation (e.g., Demeler and Saber, 1998) and is the author ofthe state-of-the-art UltraScan IIsoftware(http://www.ultrascan.uthscsa.edu).

An in vivo assay for DsRed oligomerization may be used in a geneticscreen. For example, a yeast two-hybrid system such as that reported tohave has been used to verify the oligomerization of wild-type DsRed(Baird et al., 2000) may be used to assess the tendancy of DsRedvariants to oligomerize.

Another approach used to monitor DsRed oligomerization in vivo was tofuse GFP or DsRed to the protein Gos1p in S. cerevisiae. Gos1p is amembrane protein anchored to the cytoplasmic face of yeast Golgicisternae. The GFP-Gos1p fusion protein gives a fluorescence patternthat is typical for the Golgi in S. cerevisiae, whereas a wild-typeDsRed Gos1p fusion generates large red blobs in the cells, presumablybecause multiple Golgi cisternae become crosslinked via DsRedtetramerization. Thus, visualizing DsRed-Gos1p fusions provides an invivo assay for DsRed oligomerization.

In another screening assay, randomly mutagenized DsRed proteins will befused to the C-terminus of glutathione S-transferase (GST; Smith andJohnson, 1988). Because GST is a dimer (McTigue et al., 1995), thefusion of GST to an oligomeric DsRed variant will generate crosslinkedaggregates that will be insoluble upon gentle detergent lysis of thecells. By contrast, the fusion of GST to a monomeric DsRed variant willgenerate a soluble protein. The concept of using this method to evaluatethe oligomeric state of DsRed variants was tested using an expressionand detergent lysis protocol similar to that used to evaluate solubilityor aggregation of DsRed, fluorescent DsRed.T1, a tetrameric variant, wasefficiently extracted from the bacterial cells whereas the fluorescentGST-DsRed.T1 was quantitatively retained in the pellet. It is expectedthat this assay will provide a sensitive screen for a monomeric DsRed.

Optimizing Spectral Properties

In an earlier stage of this project, we attempted to create a bright,red-shifted DsRed variant. We began with DsRed.T4 and introduced a K83Msubstitution, which had been shown to red-shift the fluorescence spectraof wild-type DsRed (Baird et al., 2000). In the DsRed.T4 background,K83M red-shifted the spectra (FIG. 2), but substantially reduced theintrinsic brightness of the protein. Screening of randomly mutagenizedvariants was undertaken to identify variants having restoredfluorescence. Surprisingly, an L174Q mutation was found to increasebrightness. Subsequent tests confirmed that in the context of K83M,other substitutions at the polar interface, including H162S and H222S,preserve or restore colony fluorescence.

Without being limited as to theory, we speculate that the K83M mutationallows DsRed to tolerate changes at the polar interface by stabilizingthe immature form of the DsRed, thereby allowing for correct folding.The cores of most proteins are hydrophobic, but in the case of DsRed,K83 is one of several charged residues that face the interior of theprotein (Wall et al., 2000; Yarbrough et al., 2001). It may be thatimmature DsRed is stabilized by tetramerization, particularly byinteractions at the polar interface, and that the K83M substitutionrenders the immature protein sufficiently stable to fold even in theabsence of interactions at the polar interface. Based on thishypothesis, we predict that in the absence of K83M, mutations such asL174Q will decrease the yield of mature DsRed but will not reduce theintrinsic fluorescence of the mature protein. Meanwhile, regardless ofthe reason, K83M has enabled us to mutagenize the polar interface.

To address the reduced fluorescence found with variants having a K83Msubstitution, two rounds of random mutagenesis and screening wereundertaken. Two additional substitutions (K163H and Y193H) thatsignificantly increase the brightness of the purified protein wereidentified. These new mutations are distinct from L174Q, and they alterresidues that face the interior of the protein. The variant of DsRed.T4having the K83M, K163H, and Y193H substitutions was designated DsRed.T6.As judged by colony fluorescence, DsRed.T6 is comparable to DsRed.T4 inbrightness. Interestingly, the spectral red-shifting observed with K83Mis largely reversed by the two additional substitutions in DsRed.T6(FIG. 2). As can be seen from FIG. 2, the K83M substitution red-shiftsthe excitation and emission peaks by ˜20 nm. The two additionalsubstitutions present in DsRed.T6 reverse most of this red-shifting, andalso suppress the green emission. We have also introduced K83M, K163H,and Y193H substitutions into the DsRed.D1 background and the resultingvariant, designated DsRed.D3, has strong fluorescence and toleratesmutations at the polar interface. We plan to start with DsRed.D3 andmutate most or all of the residues that contribute to the polarinterface.

Preferably, the monomeric DsRed variant of the present invention retainsthe spectral properties of the tetramer, namely, bright red fluorescencewith minimal green emission. However, fluorescent bacterial coloniesexpressing polypeptides containing substituted amino acids for those ofthe wild-type DsRed that ordinarily form the hydrophobic tetramerizationinterface appear somewhat dimmer than colonies of bacteria expressingtetrameric variants. Two kinds of changes may account for decreasedfluorescence of the bacterial colonies expressing mutant DsRed. First, amutation may reduce the intrinsic brightness of DsRed by lowering theextinction coefficient and/or the quantum yield. Second, a mutationmight slow DsRed maturation and/or reduce the percentage of the DsRedmolecules that eventually become fluorescent. To counteract sucheffects, random mutagenesis strategy will be used to identify brightervariants of the monomers.

Minimizing the Green Emission of Monomeric DsRed

A predicted side effect of disrupting the DsRed tetramer will be a lossof FRET and a consequent increase in the green emission. To alleviatethis problem, mutations that increase the ratio of red to greenmolecules in mature DsRed may be introduced. Substitutions of K83M,K163H, and Y193H in DsRedT6 correlate with brighter red fluorescence andreduced green emission, relative to that of the DsRed.T4+K83M variant.

In addition to mutations identified in these random screens, directedmutations aimed at weakening the tetramerization may fortuitously reducethe percentage of green molecules in mature DsRed. For instance, theA145P substitution that we incorporated into DsRed.T3 and DsRed.T4 tolower the green emission was originally generated during our attempts tomutagenize the polar interface. More recently, we found that an H222Ssubstitution at the polar interface decreases the green emission.

Screening for Reduced Blue Excitation

Monomeric variants of interest having significant green emission, may befurther mutagenized and screened for reduced green emission or reducedblue excitation. A 488-nm laser is used to excite fluorescence inbacteria containing mutant DsRed proteins, and the bacterial cells arethen sorted by flow cytometry to identify clones with reduced greenemission. This approach would be more difficult with a monomeric DsRed,because undesirable mutations that caused protein aggregation wouldsuppress the green emission due to FRET. Alternatively, mutants may bescreened for reduced blue excitation. When total fluorescence ismeasured, the signal obtained by excitation with blue light shouldcorrelate with the percentage of the DsRed molecules having a greenfluorophore, regardless of whether the emission spectrum has beenmodified by FRET.

The assay for reduced blue excitation is based on our standard slideprojector method, except that the plates will be photographed with adigital camera. We will photograph each plate under two conditions.First, the total fluorescence after excitation with blue light will berecorded by illuminating through a 485±11 nm bandpass filter andcapturing the emission signal through a Kodak Wratten filter #12, whichpasses wavelengths above 520 nm (Cronin and Hampton, 1999). The image ofthe plate will be colorized green using Adobe Photoshop. Second, thetotal fluorescence after excitation with yellow light will be recordedby illuminating through a 520±20 nm bandpass filter and capturing theemission signal through a Kodak Wratten filter #22, which passeswavelengths above 550 nm. This image of the plate will be colorized red.The red image will be due almost exclusively to DsRed molecules with thered fluorophore, whereas the green image will include a strongcontribution from DsRed molecules with the green fluorophore. Uponmerger of the red and green images, most of the colonies will appearyellow, but colonies having an increased ratio of red to green moleculeswill appear orange.

Evaluating Solubilities

One approach to evaluating solubilities of the fluorescent proteins inis as follows. E. coli cells carrying inducible expression vectorsencoding hexahistidine-tagged wild-type DsRed or DsRed variants weregrown to an optical density (OD) (λ=600 nm) of 0.5, induced for 7 h,lysed with B-PER II detergent (Pierce) and centrifuged for 20 min at27,000×g. Equivalent amounts of the pellet and supernatant fractionswere subjected to SDS PAGE followed by immunoblotting with ananti-hexahistidine antibody. The percentage of each protein in thesupernatant fraction was then quantified for each protein and thepercentage of protein molecules extracted (i.e., solubilized) wasdetermined. Typically, only about 25% of the wild-type DsRed moleculesare solubilized. Suitably, at least 30% of DsRed variant molecules aresolubilized. Preferably, at least 50% of DsRed variant molecules aresolubilized. More preferably, at least 70% of DsRed variant moleculesare solubilized.

Another approach to evaluate variants for reduced aggregation is bynondenaturing SDS-PAGE. One μg of each purified DsRed variant was mixedwith SDS-containing sample buffer on ice and immediately electrophoresedat 4° C. in a 10% polyacrylamide gel, followed by staining withCoomassie Blue. Additional aliquots of of wild-type DsRed and DsRedvariants were denatured by boiling prior to electrophoresis. Migrationof proteins as a diffuse band that may reflect the formation ofhigher-order oligomers, whereas formation of a sharp band or bands ofthe appropriate size suggests reduce aggregation.

Creation and Characterization of a Red Fluorescent DsRed Monomer

A coding sequence and the amino acid sequence of one mutant according tothe present invention, designated DsRed.M1, is shown as SEQ ID NO:4 andSEQ ID NO:5, respectively. DsRed.M1 contains the following 37substitutions relative to DsRed.T4:

A2D* V96S K163H I210V S3N T106E L174T R216H S4T T108Q V175C G219A R13QC117T E176D H222S E26Y I125K S179T L223G R36K S131A I180V F224S K47QM141A M182K L225Q V71A R149K Y192N K83M R153Q Y193H K92T H162S S203N*A2D is a second mutation of residue 2, which was an arginine inwild-type DsRed.

Oligomeric State: DsRed.M1 behaves functionally as a monomer. Theprotein appears to be monomeric as judged by nondenaturing SDS-PAGE orsize exclusion chromatography.

Brightness: The DsRed.M1 mature protein exhibits detectable redfluorescence, but is less bright than tetrameric variants, such asDsRed.T4. DsRed.M1 is also less bright than a monomeric red fluorescentDsRed protein designated mRFP1, which was generated by Tsien et al. (US2003/0170911 A1)

Spectral Properties: In contrast to mRFP1, which has a red-shift,DsRed.M1 has excitation and emission spectra similar to those of thetetrameric DsRed variants. Surprisingly, DsRed.M1 has negligible greenemission. The lack of green emission is fortunate, and ratherunexpected, given that the tetrameric DsRed variants exhibit greenemission that is largely suppressed by intersubunit resonance energytransfer.

Photostability: mRFP1 photobleaches much faster than the tetramericDsRed variants, an effect that may correlate with the altered spectralproperties of mRFP1. It is expected that DsRed.M1 will be morephotostable than mRFP1 because the spectral properties of DsRed.M1 aresimilar to those of the tetrameric variants.

Maturation: DsRed.M1 appears to retain the rapid maturation of theparental DsRed.T4, which is considerably faster than that wild-type ofDsRed.

Each publication cited in herein or in the appendix is incorporated byreference in its entirety. Also incorporated by reference in itsentirety is WO 03/054158A2.

LITERATURE CITED

-   Baird, G. S., D. A. Zacharias, and R. Y. Tsien. 2000. Biochemistry,    mutagenesis, and oligomerization of DsRed, a red fluorescent protein    from coral. Proc. Natl. Acad. Sci. USA. 97:11984–11989.-   Bogan, A. A., and K. S. Thorn. 1998. Anatomy of hot spots in protein    interfaces. J. Mol. Biol. 280:1–9.-   Cronin, S., and R. Hampton. 1999. A genetics-friendly GFP assay.    Trends Cell Biol. 9:36.-   Demeler, B., and H. Saber. 1998. Determination of molecular    parameters by fitting sedimentation data to finite-element solutions    of the Lamm equation. Biophys. J. 74:444–454.-   Gurskaya, N. G., A. F. Fradkov, A. Terskikh, M. V. Matz, Y. A.    Labas, V. I. Martynov, Y. G. Yanushevich, K. A. Lukyanov, and S. A.    Lukyanov. 2001. GFP-like chromoproteins as a source of far-red    fluorescent proteins. FEBS Lett. 507:16–20.-   Laue, T. M., and W. F. Stafford, 3rd. 1999. Modern applications of    analytical ultracentrifugation. Annu. Rev. Biophys. Biomol. Struct.    28:75–100.-   Matz, M. V., A. F. Fradkov, Y. A. Labas, A. P. Savitsky, A. G.    Zaraisky, M. L. Markelov, and S. A. Lukyanov. 1999. Fluorescent    proteins from nonbioluminescent Anthozoa species. Nat. Biotechnol.    17:969–973.-   McTigue, M. A., D. R. Williams, and J. A. Tainer. 1995. Crystal    structures of a schistosomal drug and vaccine target: glutathione    S-transferase from Schistosoma japonica and its complex with the    leading antischistosomal drug praziquantel. J. Mol. Biol. 246:21–27.-   Pierce, D. W., and R. D. Vale. 1999. Single-molecule fluorescence    detection of green fluorescence protein and application to    single-protein dynamics. In Green Fluorescent Proteins (Methods in    Cell Biology, Vol. 58). K. F. Sullivan, and S. A. Kay, editors.    Academic Press, San Diego. 49–73.-   Raffen, R., and F. J. Stevens. 1999. Small zone, high-speed gel    filtration chromatography to detect protein aggregation associated    with light chain pathologies. Methods Enzymol. 309:318–332.-   Smith, D. B., and K. S. Johnson. 1988. Single-step purification of    polypeptides expressed in Escherichia coli as fusions with    glutathione S-transferase. Gene. 67:31–40.-   Tsien, R. Y. 1998. The green fluorescent protein. Annu. Rev.    Biochem. 67:509–544.-   Wall, M. A., M. Socolich, and R. Ranganathan. 2000. The structural    basis for red fluorescence in the tetrameric GFP homolog DsRed. Nat.    Struct. Biol. 7:1133–1138.-   Yarbrough, D., R. M. Wachter, K. Kallio, M. V. Matz, and S. J.    Remington. 2001. Refined crystal structure of DsRed, a red    fluorescent protein from coral, at 2.0-Å resolution. Proc. Natl.    Acad. Sci. USA. 98:462–467.

1. An isolated polynucleotide encoding a variant polypeptide ofwild-type DsRed of SEQ ID NO: 2 or a variant of the rapidly maturingDiscosoma red fluorescent protein T4 (DsRed.T4) of SEQ ID NO: 3, thevariant polypeptide having reduced oligomerization relative wild-typeDsRed, the variant polypeptide comprising the amino acid substitutionsK83M, K163H, and Y193H, the variant polypeptide further comprising atleast one amino acid substitution selected from the group consisting ofE26Y, K92T, V96S, T106E, T108Q, I125K, S131A, I180V, and M182K thevariant polypeptide further comprising at least one amino acidsubstitution selected from the group consisting of R13Q, R36K, K47Q,M141A, and I210V.
 2. The isolated polynucleotide of claim 1, wherein thevariant polypeptide exists primarily as a monomer.
 3. An isolatedpolynucleotide encoding a variant polypeptide of the rapidly maturingDiscosoma red fluorescent protein T4 (DsRed.T4) of SEQ ID NO: 3, thevariant polypeptide exhibiting reduced oligomerization relative toDsRed.T4 and exhibiting detectable red fluorescence, the variantpolypeptide comprising: at least one amino acid substitution selectedfrom the group consisting of K83M, K83L, K163Q, K163M, K163H, and Y193H;at least three amino acid substitutions selected from the groupconsisting of E26Y, K92T, V96S, T106E, T108Q, I125K, S131A, I180V, andM182K; and at least three amino acid substitutions selected from thegroup consisting of R149K, R153Q, H162S, L174T, E176D, Y192N, R216H,H222S, L223G, and F224S.
 4. The isolated polynucleotide of claim 3,wherein the variant polypeptide further comprises at least one aminoacid substitution selected from the group consisting of A2D, S3N, andS4T.
 5. The isolated polynucleotide of claim 3, wherein the variantpolypeptide further comprises at least one amino acid substitutionselected from the group consisting of R13Q, R36K, K47Q, M141A, andI210V.
 6. The polynucleotide of claim 3, wherein the variant polypeptidefurther comprises the amino acid substitution D6N.
 7. The polynucleotideof claim 3, wherein the variant polypeptide further comprises at leastone amino acid substitution selected from the group consisting of D115G,D116N, K121H, K168E, and D169G.
 8. The polynucleotide of claim 3,wherein the variant polypeptide comprises the amino acid substitutionY193H.
 9. The polynucleotide of claim 8, wherein the variant polypeptidecomprises at least one of amino acid substitutions K83M and K163H. 10.The isolated polynucleotide of claim 9, wherein the variant polypeptidecomprises amino acid substitutions E26Y, K92T, V96S, T106E, T108Q,I125K, S131A, I180V, M182K, R149K, R153Q, H162S, L174T, E176D, Y192N,R216H, H222S, L223G, and F224S.
 11. The isolated polynucleotide of claim9, wherein the variant polypeptide comprises 95% amino acid identity tothe sequence of SEQ ID NO:
 5. 12. The isolated polynucleotide of claim11, wherein the variant polypeptide comprises SEQ ID NO:5.
 13. Theisolated polynucleotide of claim 9, wherein the variant polypeptidecomprises at least one amino acid substitution selected from the groupconsisting of D6N, D115G, G116N, K121H, K168E, and D169G.
 14. Theisolated polynucleotide of claim 3, wherein the polypeptide exists as amonomer.
 15. The isolated polynucleotide of claim 3, wherein thepolynucleotide further encodes a polypeptide of interest linked to thevariant polypeptide, the polypeptide of interest and the variantpolypeptide being expressed as a fusion protein.
 16. A cell comprisingthe sequence of claim
 3. 17. A construct comprising the polynucleotideof claim 3 operably linked to a promoter.
 18. A vector comprising theconstruct of claim
 17. 19. A method of obtaining expression of a variantpolypeptide of DsRedT4 comprising introducing the vector of claim 18into a host cell under conditions that permit expression of the variantpolypeptide.
 20. The method of claim 19, further comprising evaluatingthe expression of the variant polypeptide by detecting red fluorescence.21. The method of claim 19 further comprising monitoring temporal orspatial changes in red fluorescence.
 22. A method for obtainingexpression of a fusion protein of a variant polypeptide of DsRedT4 and aprotein of interest comprising introducing a vector comprising thepolynucleotide of claim 3 that further encodes the protein of interestfused to the variant polypeptide into a host cell under conditions thatpermit exxpression of the fusion protein.
 23. The method of claim 22,further comprising evaluating the expression of the fusion protein bydetecting red fluorescence.
 24. The method of claim 22, furthercomprising monitoring temporal or spatial changes in red fluorescence.